Police said around 45,000 people took to the streets of the Belgian capital for a rally that comes amid campaigning for a regional election in Catalonia on December 21.
Singing and chanting slogans, protesters marched around the EU's main institutions, some draped in flags or carrying signs with "Shame on You" stamped over an EU flag. A few Belgian Flemish separatists and Scottish flags could be seen in the crowd.
Spain says unilateral moves by Catalan officials to declare independence violate the Spanish constitution.
Former Catalan leader Carles Puigdemont, among those up for re-election on Dec. 21, used the gathering Thursday as part of his political campaign. He and four former Catalan government cabinet members fled to Brussels after the referendum fearing arrest. Several of their colleagues were jailed in Spain.
"Have you ever seen somewhere in the world such a demonstration that would support criminals?" Puigdemont asked the crowd.